Citrulline immunohistochemistry may not necessarily identify nitric oxide synthase activity: the pitfall of...

Nitric oxide synthase (NOS) converts L-arginine as a substrate to form nitric oxide and the "by-product" citrulline. To characterize NOS activity in the nervous tissue at the single-cell level, citrulline immunostaining is considered to be a suitable means of working on the principle that in brain tissue, due to the incomplete urea cycle, citrulline is produced exclusively by NOS. This assumption is correct for free citrulline but it does not consider the conversion of arginine to citrulline residues of proteins by the calcium-dependent peptidylarginine deiminase (PAD). Using a polyclonal antiserum against citrulline we observed in cerebellar cell cultures immunopositivity in a few, mostly NOS-positive, neurons, in activated microglia, and in oligodendroglia (which under control conditions are in doubt to be able to express NOS), but not in astroglia. Treatment with the excitotoxin kainate substantially enhanced the staining intensity for citrulline in neurons and glial cells. To distinguish between free (NOS-related) and protein-bound (PAD-related) citrulline we blocked NOS activity by 7-nitroindazole or L-N5-(1-iminoethyl)lysine. The results provide evidence that citrulline immunolabeling results partly from PAD-mediated protein citrullination, enhanced pathophysiologically under stimulated conditions by exposure to kainate. Our immunocytochemical observations were corroborated by Western blot analysis showing several bands of citrulline-positive proteins, whose number and staining intensity depended on kainate treatment and calcium ions.